A fragmented small-size antibody is a promising antibody capable of overcoming the limitations of the existing antibody therapeutics because of its physicochemical properties different from the full-length monoclonal antibody (mAb). There are a single chain antibody (scFv), a Fab (fragment antibody-binding) antibody, an immunoglobulin variable domain antibody such as VH or VL, and the like in general antibody fragments, and a tandem scFv, a diabody, a minibody, and the like in their modification forms (Better et al., Science 1988 240(4855):1041-3; Huston et al., Proc. Natl. Acad. Sci. USA. 1988 85(16):5879-83; Bird et al., Science 1988 242(4877):423-6; Pei et al., Proc. Natl. Acad. Sci. USA. 1997 94(18):9637-42; Iliades et al., FEBS Lett. 1997 409(3):437-41; Ward et al., Nature 1989 341(6242):544-6).
A antibody fragment or a small-size antibody mainly loses functions of Fc (crystallizable fragment) as compared with the full-length monoclonal antibody, and thus, there are no anticipated effects due to the existence of Fc, such as, an increased circulating half-life, an effector function, and the like.
However, the small-size fragmented antibody is being magnified as a next generation antibody capable of overcoming the limitations, such as limitations on accessibility to epitope structurally hidden, drug penetration and biodistribution, format flexibility, high production costs, and the like, which result from a large size of the existing whole antibody (Zhao et al., Blood 2007 110(7):2569-77; Holliger et al., Nat. Biotechnol. 2005 23(9):1126-36; Hudson et al., Med. Microbiol. Immunol. 2009 198(3):157-74; Enever et al., Curr. Opin. Biotechnol. 2009 20(4):405-11).
Furthermore, various kinds of antibody fragments and small-size antibodies have an advantage in that dual or multiple-specificity can be realized by connection in a chemical method or a recombinant protein fusion method.
Recently, this advantage has been utilized to introduce the multiple-specificity such that the Fc function is modularized, and thereby, supplementing the effector function and a short circulating half-life, which has been pointed out as disadvantages. For example, a fragment or a small-size antibody having binding specificity toward human serum albumin is introduced into a module to realize a dual-specificity antibody, thereby increasing the circulating half-life, or a small-size antibody having specific affinity to an immune cell, such as a natural killer or a T cell, is introduced into a module, thereby conferring a cell killing function thereto (Els et al., J Biol Chem. 2001 9; 276(10):7346-50; Bargou et al., Science 2008 321(5891):974-7; et al., Mol. Cancer Ther. 2008 7(8):2288-97). Also, since a single antibody can be designed to confer specificity to two or more molecules targets anticipating different action modes, the possibility that efficacy and economic feasibility of the antibody are significantly improved is opened.
A smallest unit of human antibody structure that has an antigen-specific binding function is a heavy chain variable domain (VH) or a light chain variable domain (VL), which is a variable domain positioned at the N-terminal of a light chain or a heavy chain. Since respective two N-terminals have been evolved to have a complementary structure, VH and VL constitute a non-covalent binding type complex in the procedure of assembling the heavy chain and the light chain when a monoclonal antibody is produced from a plasma B cell, and thereby maintain structural stability thereof. Human antibody variable domain VH segments are classified into 7 families (VH1, VH2, VH3, VH4, VH5, VH6, VH7) depending on the homology of amino acid sequences in a frame portion, excluding CDRs (complementarity determining regions) binding to epitope, and each family contains three to twenty-two kinds of distinct amino acid sequences. VLs of the light chain are divided into V kappa and V lamda, and V kappa is classified into six families and V lamda is classified into ten families (Chothia et al., 1992 J. Mol. Biol. 227, 799-917; Tomlinson et al., 1995 EMBO J. 14, 4628-4638; Williams et al., J. Mol. Biol. 264, 220-232). It has been known that a number of VH and VL have preferred VH/VL pairing combinations depending on the degree of mutual affinity, and thus, it has been known that this combinatorial rearrangement of genes has an important role in enlarging diversity of antibody repertoire (Ruud et al., J. Mol. Biol. 1999, 285, 895-901).
A bound type of VH/VL confers complementary binding specificity to a particular antigen according to combinations of 6 CDRs. CDR1 CDR2, and CDR3 of the light chain and CDR1 CDR2, and CDR3 of the heavy chain, which are a total of 6 CDRs, participate in binding to the antigen. According to analyses of human germ-line sequence, it was found that a variety of the respective CDRs mostly depend on CDR3 of the heavy chain variable domain. Therefore, this analysis implies that the binding specificity of an antibody mostly depends on variability of the heavy chain CDR3 (J. Mol. Recogni. 2000, 13, 167-187).
Unlike this, animals such as a camel and a llama and fish having a cartilage backbone such as a shark have antibodies of a single heavy chain structure without a light chain structure. Therefore, a variable domain of theses antibodies include only a single heavy chain variable domain (VHH and VNAR for camel and shark, respectively), and it is known that this antibody is no less competent than the human antibody where VH and VL simultaneously participate in binding to antigens, in view of binding to antigens and a neutralizing function. VH or VL alone is rarely present, except in human patients having heavy chain diseases (Hendershot et al., J. Cell. Biol. 1987 104(3):761-7; Prelli et al., J. Immunol. 1992 148(3): 949-52). The reason is that VH or VL is not structurally stable at the time of separation of VH or VL alone due to structural complementarity thereof, and thus, protein aggregation may easily occur. It is known that this protein aggregation partially results from hydrophobic interaction caused by distribution of hydrophobic amino acid residues mainly at an interface of VH and VL. In the case of camel antibody, amino acid residues having hydrophilicity may be specifically positioned on the surface of VH/VL border region, unlike human antibody. Particularly, amino acids at four sites of the camel antibody, which are specifically different from those of a human VH3 family, are called a tetrad. These amino acids are positioned at 37, 44, 45, and 47 in a Kabat numbering system (Kabat et al., 1991 J. Immunol. 147(5), 1709-1719). This difference in the amino acid sequence may explain stability of a single variable domain antibody (VHH). There was an attempt to produce improved, camelid antibodies by replacing amino acids at tetrad positions with hydrophilic amino acids of the camel antibody (G44E/L45R/W47G) in the human variable domain antibody.
As a result, solubility thereof may be somewhat improved in view of physical and chemical properties (Coppieters et al., Arthritis Rheum. 2006 54(6):1856-66; Dolk et al., Proteins. 2005 59(3):555-64; Ewert et al., Biochem. 2002 41(11):3628-36; Kortt et al., J. Protein Chem. 1995 14(3):167-78; Martin et al., Protein Eng. 1997 10(5):607-14). However, stability thereof is difficult to obtain as compared with the camel antibody, for example, decreased protein expression yield and thermostability (Davies et al., FEBS Lett. 1994 Feb. 21; 339(3):285-90; Aires et al., J. Mol. Biol. 2004 340(3):525-42). It has been found that the reason therefor was that modification of amino acids at the VH/VL border region causes modification in a beta-sheet structure of the corresponding region (Riechmann et al., J. Mol. Biol. 1996 259(5):957-69). The CDR3 of the camel single domain antibody has an abnormally long loop structure as compared with the human antibody. According to structural analysis, it was found that this loop structure folds into the VH/VL border region of the human antibody, and it has been suggested that this distinct structure partially shields a hydrophobic patch positioned at the border region, thereby helping stabilization of the camel single domain antibody (Joost et al., 2010 Drug Discovery Today: Technologies 7(2), 139-146).
This shielding effect is hardly anticipated in the human antibody due to a relatively short loop structure of CDR3. In conclusion, the human single variable domain itself has deficient physical and chemical properties as compared with the camel single domain antibody, and thereby is not sufficient to be utilized as a scaffold of a binding ligand to a particular antigen. As the method for overcoming this, mere replacement of tetrad amino acids which are structural signatures of the camel antibody is not sufficient, and protein structure design and directed evolution of VH or VL are further needed.
A human immunoglobulin variable domain (VH or VL) that exists in nature is a minimum-size antibody ( 1/12 the size of monoclonal antibody) capable of maintaining an antigen binding characteristic, and thus, is anticipated to be different from the conventional monoclonal antibody in view of physical properties and therapeutic effects as a therapeutic protein. Hence, a demand for developing human antibodies having only one variable domain has increased. Nevertheless, aggregation and unstable tendency of protein when VH or VL alone exists still remain as major obstacles that should be overcome in developing a binding scaffold with respect to a specific antigen.
Accordingly, in order that an antibody fragment and a small-size antibody provide advantages that cannot be achieved by general monoclonal antibodies and stay competitive themselves, it is important to secure robust pharmaceutical and physicochemical properties of a substance itself.
Some molecular directed evolution methods have been attempted also in the prior art so as to stabilize human heavy chain or light chain variable domains (Barthelemy et al., J. Biol. Chem. 2008 283(6):3639-54). They constructed a phage display system with a CDR-engineered library of VH, and then screened VHs having binding activity toward the protein A after applying thermal stress. There was a report that CDR engineered human VH having increased solubility, and allowing reversible folding after protein thermal denaturation was screened by this method (Jespers et al., Nat. Biotechnol. 2004 22(9):1161-5). Also, there was a report that various libraries where mutations were induced at a CDR3 portion and a frame portion without thermal denaturation treatment were prepared, and VH exhibiting high binding activity toward protein A after phage display was screened by the same method, and thus, an engineered VH that is thermodynamically stable and has increased soluble expression as compared with a wild type VH can be obtained (Barthelemy et al., J Biol Chem. 2008 283(6):3639-54). In the phage display system, the target protein is induced to a Sec pathway by a Sec signal sequence of pelB protein fused to N-terminal of the target protein. However, in this case, the protein, which is previously folded within the cytoplasm of E. coli cannot pass through the pathway due to the limitation of an inherent translocation pathway of a protein. The reason is that a general phage display uses a Sec pathway, which is a representative protein translocation pathway of E. coli, and, due to the nature of this pathway, target protein has a linear structure not a three-dimensional structure with the help of chaperon within the cytoplasm when passing through a cell membrane. Sec pathway-specific proteins that naturally exist, distinctively in a linear form, without being folded, with the help of a chaperon called SecB within the cytoplasm, directly after protein transcription. The Sec pathway target protein, which is moved to a translocase complex, consisting of Sec A, SecYEG, and SecDFYajC existing on an intracellular membrane by Sec B, passes through the membrane in a linear form, without being in a three-dimensional structure, and the passed amino acid chain forms a complete three-dimensional structure, including a disulfide linkage, by oxidation and reduction of DsbA and DsbB until it arrives at the periplasm (Baneyx and Mujacic Nature Biotech. 2004, 22, 1399˜1408). Therefore, if folding and three-dimensional structure formation of certain a protein quickly occurs in the cytoplasm due to the nature of the protein itself, this protein does not have compatibility with a phage display screening system designed to the Sec pathway.
In addition, it was reported that the wild type VH having improved physiochemical properties could be selected when clones are directly screened from a plate spread with bacterial lawn based on the size of a plaque size (To et al., J. Biol. Chem. 2005 280(50):41395-403). However, large-scale treatment is impossible by plate-based screening, and thus, in order to reduce the size of the library, an initial library for only the VH3 family subjected to a protein A screening procedure in vitro was manufactured.
Meanwhile, in order to improve folding characteristics of the recombinant protein, a genetic selection method was attempted (Maxwell et al., Protein Sci. 1999 8(9):1908-11; Wigley et al., Nat. Biotechnol. 2001 19(2):131-6; Cabantous et al., Nat Biotechnol. 2005 23(1):102-7; Waldo G S. Curr. Opin. Chem. Biol. 2003 7(1):33-8). One of the representative methods for improving folding characteristics of the recombinant protein is that the folding degree of a protein of interest is indirectly determined by measuring activity of a reporter protein fused to the protein of interest in a recombinant DNA technology. However, the folding cannot be accurately reflected when the protein of interest exists alone.
In addition, in order to increase solubility of the protein, there has been developed a molecular directed evolution method where a Tat (twin-arginine translocation) pathway, which is a protein tranlocation pathway having a function of proof-reading folding quality of proteins, is utilized as a biological filter of determining whether or not the protein is folded. Specifically, the protein of interest is fused to a reporter gene and a Tat signal sequence and expressed by the Tat pathway within Escherichia coli, and then is subjected to a protein folding proof-reading by a Tat ABC translocase complex according to folding degrees and solubility of the protein. If the target protein has sufficient solubility, a fusion protein consisting of the target protein and the reporter protein passes through an inner membrane of Escherichia coli and reaches the periplasm. The fusion protein reaching the periplasm is detected by a method such as antibiotic resistance measurement or the like, thereby screening proteins having a desired degree of solubility (Fisher et al., Protein Sci. 2006 15(3):449-58). It can be seen that, when the recombinant protein, not only a Tat pathway substrate protein in a natural system, is applied to the Tat pathway, but it also significantly passes through the Tat pathway in proportional to solubility and stability of recombinant proteins (Lim et al., Protein Sci. 2009 18(12):2537-49). In addition, it has been reported that a single chain antibody (scFv) allowing protein folding within the cytoplasm of E. coli is effectively screened by using the Tat pathway (Fisher A C and DeLisa M P. J Mol Biol. 2009 385(1): 299-311). According to the above document, molecular directed evolution was completely achieved in vitro by using scFv13 that is insoluble in and expressed in E. coli as a template base sequence. A disulfide bond presents within scFv has a level of about 4 to 6 kcal/mol, and contributes to stabilization of protein molecules. This bond is formed in an oxidizing environment such as the periplasm of bacteria or the endoplasmic recticulum (ER) of eukaryote. The periplasm of bacteria usually maintains oxidation conditions through the flow of electrons between DsbA and DsbB present on an inner membrane. Therefore, in the case of scFv13 protein selected from the scFv-engineered library by artificially passing through the Tat pathway, intrabodies, which are autonomously self-folded without forming a disulfide bond within reduction conditional cytoplasm but not oxidation conditional cytoplasm, are preferentially selected. Specifically, when a gene where a signal sequence leading the protein to the Tat pathway is fused to N-terminal of the target protein and TEM-1 beta-lactamase is fused to a C-terminal of the target protein, for functioning as a reporter gene, is expressed within E. coli, a triple-function fusion protein (tripartide) is expressed.
The expressed fusion protein heads for the Tat pathway, and is subjected to a protein folding inspection by machinery of a Tat ABC translocase complex existing on the inner cell membrane. Several studies found that, among many recombinant proteins, only those having solubility keeps compatibility with specific machineries of the Tat pathway (Sanders et al., Mol. Microbiol. 2001 41(1):241-6; DeLisa et al., Proc. Natl. Acad. Sci. USA. 2003 100(10):6115-20; Matos et al., EMBO J. 2008 27(15):2055-63; Fisher A C and DeLisa M P. J. Mol. Biol. 2009 385(1): 299-311; Lim et al., Protein Sci. 2009 18(12): 2537-49).
However, the above method for improving the folding characteristics of protein has been never applied in selecting domain antibodies, particularly, VH or VL domain antibodies or the like.
In conclusion, presently, engineered modification and screening of human VH domain antibody were unexceptionally conducted based on phage display and binding activity to protein A (Kristensen P and Winter G. Fold. Des. 1998 3(5):321-8; Sieber et al., Nat. Biotechnol. 1998 16(10):955-60; Jung et al., J. Mol. Biol. 1999 294(1):163-80; Wörn A and Plückthun A. J. Mol. Biol. 2001 305(5): 989-1010).
Therefore, methods of selecting VH domain antibodies having more efficient solubility and high thermostability are desperately in need of development, and further, the smallest unit next generation antibodies having improved efficacy by utilizing the selected domain antibodies are promptly in need of development.